The proposed studies are aimed at the pathway by which thrombin initiates fibrinolysis. The first step is the interaction of thrombin with an endothelial cell membrane protein, thrombomodulin (TM), which induces activity of the enzyme to activate protein C, while disabling clotting activity. A major effort will focus on the structural features of thrombin that are involved in the interaction with TM and the interactions of thrombin-TM with thrombin substrates and inhibitors. The primary tools will be thrombin derivatives that have altered affinities for TM or altered function when bound, and antibodies to thrombin that differentially influence the interactions of thrombin with its ligants. A second effort will be the initiation of primary structural analysis of TM. Large fragments will be prepared by using proteases of restricted specificities and the fragments will be resolved by electrophoresis. The fragments then will be sequenced by use of gas phase microsequencing technology. The objective of these efforts is an understanding of the chemical mechanism by which TM changes the specificity of thrombin. The next step in the thrombin-initiated fibrinolytic pathway is the sitmulation by activated protein C of the release into the circulation of the tissue type, or fibrin-dependent plasminogen activator. Cultured cells, perfused organs and intact animals will be employed to develop bioassays for humoral factors that stimulate plasminogen activator release. The bioassays will be used to determine if the stimulus is direct, that is, a primary effect of activated protein C, of if activated protein C evokes secretion of an intermediate messenger. If an intermediate messenger is involved, then the bioassays will be used to guide identification and characterization of the substance. Thrombin-initiated control of coagulation and fibrinolysis is likely to be a key pathway for regulation or confinement of hemostasis. Because the pathway contains many components, a corresponding number of derangements may on one hand provoke the development of thrombotic disease, while offering on the other hand a variety of avenues for pharmacologic manipulation.